miR-16-2 is overexpressed in PV CD34⁺ cells and contributes to abnormal erythropoiesis. (A) Levels of miR-16 were measured during induced erythroid differentiation of control (black line) and PV (red line) CD34⁺ cells. A 2-phase liquid culture system was used in which erythropoietin (EPO) was added on day 6 of culture (phase 2). Data were normalized to the mean miR-16 level measured on day 0 in the CD34⁺ cells of each patient group, which comprised 7 PV patients and 5 healthy subjects, and were expressed as percent variation. **P < .01 or greater in PV vs control cells. (B) Levels of mature miR-16 were measured in CD34⁺ cells from PV (n = 75), essential thrombocythemia (ET; n = 10), or PMF (n = 25) patients by RTQ-PCR and normalized (2^−ΔΔCT; expressed as relative quantity [RQ]) to control CD34⁺ cells (n = 10; upper and lower limits of control subjects are indicated by dashed lines). Also included were subjects with reactive erythrocytosis (RE; n = 3), hemolytic anemia (HA; n = 2), and low-risk myelodysplastic syndromes (MDS; n = 5); because all of these subjects had levels within the normal range, they have been grouped together in the plot. (C) PV CD34⁺ cells were transfected with siRNA against pre–miR-16-1 or pre–miR-16-2, and levels of mature miR-16 were measured 24 hours later; an aliquot of cells transfected with scramble (Scr) siRNA served as control. **P < .0001. (D) CD34⁺ cells from healthy subjects were transfected with pre–miR-16 (+) or scramble pre-miR (−) and plated in semisolid medium in the presence of an optimal cytokine cocktail that included EPO to permit the growth of erythroid (CFU-E, BFU-E) or myeloid (CFU–granulocyte macrophage [CFU-GM]) progenitors. Pre–miR-16 transfection rates were assessed by FACS analysis and were in the range of 60% to 70%; cultures were established with unsorted cells. Data shown were generated from 6 controls in 3 independent experiments. *P < .05; **P < .01. (E) The effect of pre–miR-16 transfection on the expression of erythroid differentiation markers was measured in a 2-phase liquid culture system that had been supplemented (“with”) or not (“w/o”) EPO on day 6. These cultures were established with pre–miR-16–transfected, unsorted, normal CD34⁺ cells (n = 5 subjects); transfection rates assessed by FACS analysis were in the range of 60% to 65%. Flow cytometric analysis for membrane erythroid marker expression was performed 4 days after EPO supplementation. *P < .05; **P < .01. (F) CD34⁺ cells from PV patients were transfected with siRNA against pre–miR-16-1 or pre–miR-16-2 and the growth of EECs, BFU-E, and CFU-GM was evaluated. **P < .01. (G) PV CD34⁺ cells were transfected with siRNA against pre–miR-16-1 or pre–miR-16-2, sorted by flow cytometry, and then plated for EEC growth. **P < .01. Representative images of EECs obtained by plating PV CD34⁺ cells that had been transfected with siRNA against pre–miR-16-1 or pre–miR-16-2 are presented on the right. Microphotographs were obtained with a Nikon Eclipse TS100 contrast-phase microscope, objective Nikon plan 40×/0.65.